Adult Respiratory Distress Syndrome (ARDS) is a serious pathological condition resulting in pulmonary failure, and therefore requiring patient hospitalization and admission to the critical care unit (1, 2). ARDS can be triggered by a variety of pulmonary and non-pulmonary causes, such as pneumonia, aspiration of gastric content and lung contusion, but also sepsis, burn, multiple trauma, and acute pancreatitis (3). ARDS is also the proximal cause of morbidity in a large percentage of patients who succumb to upper respiratory tract infections such as Severe Acute Respiratory Syndrome (SARS) caused by coronavirus SARS-CoV and Middle East Respiratory Syndrome (MERS) thought to be caused by coronavirus MERS-CoV.
Pathogenesis of ARDS includes inflammation of the lung parenchyma, infiltration of neutrophils into the airspaces, oxidative stress, disruption of the endothelial and epithelial barriers, damage to the epithelial lining and subsequent lung fibrosis. Despite the fact that the mechanisms contributing to the pulmonary failure are well delineated, more than 20 years of clinical trials show that approaches aiming at the separate components of pathogenesis fail to improve mortality (4). As of now, treatment remains primarily supportive and consists of the patient oxygenation with the lung protective ventilation strategy, and the tight control over the patient's fluid balance. Failure to alleviate ARDS with the numerous pharmacological and non-pharmacological strategies shaped a request for a novel complex therapy, which would not only limit the pathogenic mechanisms of ARDS but also facilitate lung repair (5, 6). Stem cell therapy seems to address this request for a multi-directional therapeutic action, as stem cells were shown to exert both anti-inflammatory and pro-angiogenic activity.
Some embodiments of the present disclosure include methods of treating ARDS in vivo by intravenously administering Adult Stem Cell Condition Media (ASC-CM). In some embodiments treating a patient in need thereof with ASC-CM alleviates ARDS in vivo, and reduces the mortality of currently incurable (as well as curable) diseases with significant mortality due to ARDS, caused by respiratory infections such as MERS/SARS. In some embodiments ASC and or ASC-CM and their effects include the limitation of acute kidney injury which may occur in MERS/SARS due to cytokine storm as well as hypoxia due to ARDS itself. In endotoxin-induced model of ARDS, it has been shown that ASC as well as ASC-CM diminishes lung histopathologic changes, namely the extravasation of neutrophils (inflammation) and Red Blood Cells (hemorrhage) into lung parenchyma and airspaces, and thickening of the alveolar wall. ASC-CM also inhibits endotoxin-induced increase in protein content in bronchoalveolar lavage, indicative of epithelial barrier dysfunction. It reduces endotoxin-induced release of pro-inflammatory cytokine Tumor Necrosis Factor alpha. It compromises the ability of lavage neutrophils to generate Reactive Oxygen Species, harmful to endothelial and epithelial barrier, and epithelial cell viability. In vitro, ASC-CM strengthens the ability of endothelial monolayers to counteract peroxide-induced barrier dysfunction. These data taken together clearly indicate that ASC-CM suppresses inflammation and barrier hyperpermeability, two key pathological mechanisms contributing to the development of lung edema in ARDS.
In some embodiments, ASC isolated from subcutaneous fat of the patient (frozen or freshly isolated) are expanded in the tissue culture flask/dish. ASC are grown until 30-90% confluent and incubated with growth media or basal media for 24-72 h. Collected media can be used as is, frozen, or concentrated using 3 kDa or higher cut-off filters. It has been found that the fractions larger than 50-100 kiloDaltons possess the greatest amount of activity, which is consistent with large molecules/complexes, or indeed exosomes bearing the therapeutic effect. In some embodiments, conditioned media (“CM”) is obtained and manipulated (in some embodiments fractionated) under sterile conditions to allow injection into patients with lifer-threatening manifestation of MERS/SARS.
In some embodiments, ASC-CM was tested in a mouse model of endotoxin/LPS-induced ARDS. In this model, LPS was instilled directly into lungs to mimic gram-negative pneumonia. ASC-CM was injected intravenously 4 h after LPS instillation. At this time point, mice experienced hypothermic shock, and their lungs already showed the signs of neutrophil infiltration (inflammation). This model allowed for determining the effect of ASC-CM on ongoing ARDS development. At both 24 and 48 h post-injection, histopathologic changes of the lung are markedly suppressed in mice which received ASC-CM. At 48 h, the effect on protein extravasation in bronchoalveolar lavage, and tumor necrosis factor alpha content was evident. Lavage neutrophils from mice receiving ASC-CM displayed lessen ROS generation in response to LPS. In vitro data showed that pre-incubation of endothelial monolayers with ASC-CM suppresses peroxide-induced barrier dysfunction. One of skill in the art could test ASC-CM in a rodent models of lung contusion-induced ARDS, as well as infectious models related to MERS. The findings may be reproducible in an ovine model of LPS-induced ARDS.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein ARDS is caused by an upper respiratory tract infection caused by coronavirus SARS-CoV or by coronavirus MERS-CoV.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein ARDS is caused by an upper respiratory tract infection caused by coronavirus SARS-CoV or by coronavirus MERS-CoV, where the therapeutically effective dose of ASC or ASC-CM is administered intravenously.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the patient is administered a therapeutically effective dose of ASC-CM, the ASC-CM comprising material have a molecular weight of greater than about 10,000 Daltons, 20,000 Daltons, 30,000 Daltons, 40,000 Daltons, 50,000 Daltons, 60,000 Daltons, 70,000 Daltons, 80,000 Daltons, 90,000 Daltons, 100,000 Daltons, or 150,000 Daltons.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the patient is administered a therapeutically effective dose of a fraction of ASC-CM, comprised of exosomes.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the patient is administered a therapeutically effective dose of a fraction of ASC-CM, comprised of exosomes concentrated by any of filtration, centrifugation, or precipitation and resuspension.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the patient is administered a therapeutically effective dose of a fraction of ASC-CM, comprised of exosomes, wherein the ASC-CM is administered intravenously or by aspirating the material into at least one lung of the patient.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the patient is administered a therapeutically effective dose of a fraction of ASC-CM, comprised of exosomes concentrated by any of filtration, centrifugation, or precipitation and resuspension, wherein the ASC-CM is administered intravenously or by aspirating the material into at least one lung of the patient.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the ASC-CM comprising material have a molecular weight of greater than about 10,000 Daltons, 20,000 Daltons, 30,000 Daltons, 40,000 Daltons, 50,000 Daltons, 60,000 Daltons, 70,000 Daltons, 80,000 Daltons, 90,000 Daltons, 100,000 Daltons, or 150,000 Daltons is administered intravenously.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the ASC-CM comprising material have a molecular weight of greater than about 10,000 Daltons, 20,000 Daltons, 30,000 Daltons, 40,000 Daltons, 50,000 Daltons, 60,000 Daltons, 70,000 Daltons, 80,000 Daltons, 90,000 Daltons, 100,000 Daltons, or 150,000 Daltons is administered by aspirating the material into at least one lung of the patient.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the ASC-CM comprising material have a molecular weight of greater than about 10,000 Daltons, 20,000 Daltons, 30,000 Daltons, 40,000 Daltons, 50,000 Daltons, 60,000 Daltons, 70,000 Daltons, 80,000 Daltons, 90,000 Daltons, 100,000 Daltons, or 150,000 Daltons is administered intravenously, wherein the dose of ASC-CM administered intravenously is about 0.1 ml/kg to about 2.0 ml/kg, of 100× concentrate of ASC-CM, preferably about 0.5 ml/kg to about 1.0 ml/kg, of 100× concentrate of ASC-CM.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the patient is administered a therapeutically effective dose of a fraction of ASC-CM, comprised of exosomes, wherein the dose of ASC-CM administered intravenously is about 0.1 ml/kg to about 2.0 ml/kg, of 100× concentrate of ASC-CM, preferably about 0.5 ml/kg to about 1.0 ml/kg, of 100× concentrate of ASC-CM.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the patient is administered a therapeutically effective dose of a fraction of ASC-CM, comprised of exosomes concentrated by any of filtration, centrifugation, or precipitation and resuspension, wherein the dose of ASC-CM administered intravenously is about 0.1 ml/kg to about 2.0 ml/kg, of 100× concentrate of ASC-CM, preferably about 0.5 ml/kg to about 1.0 ml/kg, of 100× concentrate of ASC-CM.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the dose of ASC administered intravenously is about 1×106 cells/kg to about 1×108 cells/kg, preferably about 1×107 cells/kg.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein ARDS is caused by an upper respiratory tract infection caused by coronavirus SARS-CoV or by coronavirus MERS-CoV, wherein the dose of ASC administered intravenously is about 1×106 cells/kg to about 1×108 cells/kg, preferably about 1×107 cells/kg.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the patient is administered a therapeutically effective dose of ASC-CM, the ASC-CM comprising material have a molecular weight of greater than about 10,000 Daltons, 20,000 Daltons, 30,000 Daltons, 40,000 Daltons, 50,000 Daltons, 60,000 Daltons, 70,000 Daltons, 80,000 Daltons, 90,000 Daltons, 100,000 Daltons, or 150,000 Daltons, further comprising the step of formulating ASC-CM material to avoid heat sensitivity.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the patient is administered a therapeutically effective dose of ASC-CM, the ASC-CM comprising material have a molecular weight of greater than about 10,000 Daltons, 20,000 Daltons, 30,000 Daltons, 40,000 Daltons, 50,000 Daltons, 60,000 Daltons, 70,000 Daltons, 80,000 Daltons, 90,000 Daltons, 100,000 Daltons, or 150,000 Daltons, further comprising the step of formulating ASC-CM material to avoid exosome sensitivity.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the patient is administered a therapeutically effective dose of a fraction of ASC-CM, comprised of exosomes, further comprising the step of formulating ASC-CM material to avoid heat sensitivity.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the patient is administered a therapeutically effective dose of a fraction of ASC-CM, comprised of exosomes, further comprising the step of formulating ASC-CM material to avoid exosome sensitivity.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the patient is administered a therapeutically effective dose of a fraction of ASC-CM, comprised of exosomes concentrated by any of filtration, centrifugation, or precipitation and resuspension, further comprising the step of formulating ASC-CM material to avoid heat sensitivity.
Some embodiments include methods for treating a patient, comprising the step of: administering at least one therapeutically effective dose of ASC or ASC-CM to a patient, wherein the patient is afflicted with ARDS and wherein the patient is a human or an animal, wherein the patient is administered a therapeutically effective dose of a fraction of ASC-CM, comprised of exosomes concentrated by any of filtration, centrifugation, or precipitation and resuspension, further comprising the step of formulating ASC-CM material to avoid exosome sensitivity.